Laser Scanning Imaging System in Support of Single-Molecule Studies of Genome Integrity

项目总结 我们的染色体不断受到各种侮辱的轰炸，造成的损害必须修复。 细胞已经进化出检测和修复断裂DNA链的机制，从而防止重要的 遗传信息。双链DNA断裂(DSB)是一种导致特别是 灾难性的后果。如果不加以纠正，DSB可能会导致严重的染色体重排，这是 是各种癌症的标志。 同源重组(HR)是细胞修复双链断裂的一条保守途径，是必需的 以防止和修复在DNA复制过程中出现的损伤。当DSB发生时，DNA末端是 加工以产生3‘单链DNA(SsDNA)突出物。单链DNA末端然后与同源序列配对 基因组中其他地方的序列，并使用同源DNA作为模板来替换缺失的DNA 用于复制。最后，复制的中间体被分解，重新获得断裂DNA的连续性。人力资源 需要一系列复杂的蛋白质的协调作用，这些蛋白质负责感知损伤， 招募要素，处理和修复受损的DNA。扰乱的后果 人力资源是毁灭性的。例如，RAD51重组酶的突变在小鼠中是胚胎致死的，以及 人类RAD51基因突变与乳腺癌有关。此外，BRCA2中的缺陷至少占5% 在所有乳腺癌中，也有卵巢癌的遗传易感性。BRCA2被认为是有帮助的 调节HR，而这一调节的缺失可能是该基因与遗传性癌症相关的原因。新的 这些发现将是充分理解这些结果的机制基础所必需的。 我们的研究计划专注于了解蛋白质如何感知和响应受损的DNA，以及它们 然后修复受损的DNA，以防止可能导致癌症的突变。为了帮助解决这些问题，我们 已经开发出独特的技术，使我们能够通过使用 光学显微镜，它使我们能够监测DNA修复和DNA的空间和时间进程 在单分子水平上实时复制。使用这种方法，我们试图定义基本的 我们的细胞用来复制和修复DNA的机制，其长期目标是了解错误是如何 在这些过程中会导致染色体重排。

PROJECT SUMMARY Our chromosomes are constantly bombarded with a variety of insults, resulting in damage that must be repaired. Cells have evolved mechanisms to detect and repair broken strands of DNA, thereby preventing loss of important genetic information. Double-stranded DNA breaks (DSBs) are a type of damage that lead to particularly disastrous outcomes. If not corrected, DSBs can cause gross chromosomal rearrangements, which are the hallmark of all forms of cancer. Homologous recombination (HR) is a conserved pathway that cells can use to repair DSBs, and HR is necessary to prevent and repair the damage that arises during DNA replication. When a DSB occurs, the DNA ends are processed to generate 3’ single-strand DNA (ssDNA) overhangs. The ssDNA ends then pair with homologous sequence elsewhere in the genome, and the missing DNA is replaced using the homologous DNA as a template for replication. Finally, the replicated intermediate is resolved, regenerating the continuity of the broken DNA. HR requires the coordinated action of a complex repertoire of proteins, which are responsible for sensing damage, recruiting essential factors, and processing and repairing the damaged DNA. The consequences of disrupting HR are devastating. For example, mutations in the RAD51 recombinase are embryonic lethal in mice, and mutations in human RAD51 are linked to breast cancers. In addition, defects in BRCA2 account for at least 5% of all breast cancers and also confer a genetic predisposition to ovarian cancer. BRCA2 is thought to help regulate HR, and loss of this regulation may be the reason why this gene is linked to hereditary cancers. New discoveries will be necessary to fully understand the mechanistic basis for these outcomes. Our research program is focused on understanding how proteins sense and respond to damaged DNA and they then repair the damaged DNA to prevent mutations that can lead to cancer. To help address these problems we have developed unique technologies that allow us to directly visualize hundreds of individual molecules using optical microscopy, which enables us to monitor the spatial and temporal progression of DNA repair and DNA replication in real-time at the single-molecule level. Using this approach, we seek to define the fundamental mechanisms that our cells use to replicate and repair DNA, with the long-term goal of understanding how errors during these processes can lead to chromosomal rearrangements.